Devices and methods for sample characterization

ABSTRACT

Devices and methods for characterization of analyte mixtures are provided. Some methods described herein include performing enrichment steps on a device before expelling enriched analyte fractions from the device for subsequent analysis. Also included are devices for performing these enrichment steps.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.15/363,908, filed Nov. 29, 2016, and entitled Devices and Methods forSample Preparation, which is a non-provisional of and claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No.62/260,944, filed Nov. 30, 2015, and U.S. Provisional Patent ApplicationNo. 62/338,074, filed May 18, 2016, each entitled “Devices, Methods, andKits for Sample Characterization,” the disclosure of each of which ishereby incorporated by reference in its entirety.

BACKGROUND

Some embodiments described herein relate to devices and methods forsample characterization and various uses thereof.

Separation of analyte components from a more complex analyte mixture onthe basis of an inherent quality of the analytes, and providing sets offractions that are enriched for states of that quality is a key part ofanalytical chemistry. Simplifying complex mixtures in this mannerreduces the complexity of downstream analysis. It can be advantageous toperform two or more enrichment steps that are orthogonal, (e.g., basedon different and/or unrelated qualities). In many cases, however, theprocess of performing orthogonal enrichment steps using known methodsand/or devices is cumbersome, and can dilute the analyte beyond thesensitivity of the downstream analytical equipment. In addition,complications can arise when attempting to interface known enrichmentmethods and/or devices with analytical equipment and/or techniques.

Methods have been used to interface protein sample preparationtechniques with downstream detection systems such as mass spectrometers.A common method is to prepare samples using liquid chromatography andcollect fractions for mass spectrometry (LC-MS). This has thedisadvantage of requiring protein samples to be digested into peptidefragments, leading to large number of sample fractions which must beanalyzed and complex data reconstruction post-run. While certain formsof liquid chromatography can be coupled to a mass spectrometer, forexample peptide map reversed-phase chromatography, these knowntechniques are restricted to using peptide fragments, rather than intactproteins, which limit their utility.

Another method to introduce samples into a mass spectrometer iselectrospray ionization (ESI). In ESI, small droplets of sample andsolution at a distal end of a capillary or microfluidic device areionized to induce an attraction to the charged plate of a massspectrometer. The droplet then stretches in this induced electric fieldto a cone shape (“Taylor cone”), which then releases small droplets intothe mass spectrometer for analysis. Typically, this is done in acapillary, which provides a convenient volume and size for ESI.Capillaries however, provide a linear flow path that does not allow formulti-step processing.

Other work has been pursued with microfluidic devices. Microfluidicdevices may be produced by various known techniques and provide fluidicchannels of defined width that can make up a channel network designed toperform different fluid manipulations. These devices offer an additionallevel of control and complexity than capillaries. In connection withESI, known devices include outwardly tapered tips and conductive edgesin an attempt to enhance the ESI in these devices. The outward taper ofknown microfluidic devices used for ESI, however, exposes the fragileTaylor cone structure to potential disturbances by turbulent air flowand results in a contact surface geometry that will support only alimited range of cone radii, which limits control over the volumeintroduced to the mass spectrometer through ESI. Additionally,electrolysis of water at the conductive edge can lead to gas bubbleformation, which interferes with the cone development.

One application for protein mass spectrometry is for characterizationduring the development and manufacturing of biologic and biosimilarpharmaceuticals. Biologics and biosimilars are a class of drugs whichinclude, for example, recombinant proteins, antibodies, live virusvaccines, human plasma-derived proteins, cell-based medicines,naturally-sourced proteins, antibody-drug conjugates, protein-drugconjugates and other protein drugs.

Regulatory compliance demands that biologics require extensive testingduring development and manufacture that is not required for smallmolecule drugs. This is because the manufacture of biologics has greatercomplexity due to, for example, using living material to produce thebiologic, greater complexity of biologic molecule, greater complexity ofthe manufacturing process. Characteristics required to be definedinclude, for example, charge, efficacy, hydrophobic changes, mass, andglycosylation. Currently these tests are done independent of each otherleading to a very time consuming and expensive process of characterizingbiologics.

SUMMARY

Some embodiments described herein relate to devices and methods that canenable the analysis of analytes in an analyte mixture. For example, manyspecific characterizations of biologic proteins are required byregulatory agencies. Methods and devices described herein can besuitable for characterizing proteins and/or other analytes. In someembodiments, methods and devices described herein can relate tocharacterizing an analyte mixture that includes one or more enrichmentsteps performed to separate an analyte mixture into enriched analytefractions.

In some instances, these analytes can be, for example, glycans,carbohydrates, DNA, RNA, intact proteins, digested proteins,antibody-drug conjugates, protein-drug conjugates, peptides, metabolitesor other biologically relevant molecules. In some instances, theseanalytes can be small molecule drugs. In some instances, these analytescan be protein molecules in a protein mixture, such as a biologicprotein pharmaceutical and/or a lysate collected from cells isolatedfrom culture or in vivo.

Some embodiments described herein can include a first enrichment step,in which fractions containing a subset of the analyte molecules from theoriginal analyte mixture are eluted one fraction at a time; theseenriched analyte fractions are then subjected to another enrichmentstep. At the final enrichment step, the enriched analyte fractions areexpelled for further analysis.

In some embodiments, one or more of the enrichment steps will besolid-phase separations. In some embodiments, one or more of theenrichment steps will be solution-phase separations.

In some embodiments, a final step concentrates the enriched analytefractions before expulsion.

In some embodiments, substantially all of the enriched analyte fractionsfrom the final enrichment step are expelled in a continuous stream. Insome embodiments, a portion of the analyte mixture (e.g., a fraction ofinterest) will be expelled from a microfluidic device via an outletconfigured to interface with an analytical instrument, such as a massspectrometer or another device configured to fractionate and/or enrichat least a portion of the sample. Another portion of the analyte mixture(e.g., containing fractions other than the fraction of interest) can beexpelled via a waste channel.

In some embodiments, the expulsion is performed using pressure, electricforce, or ionization, or a combination of these.

In some embodiments, the expulsion is performed using electrosprayionization (ESI) into, for example, a mass spectrometer. In someembodiments a sheath liquid is used as an electrolyte for anelectrophoretic separation. In some embodiments, a nebulizing gas isprovided to reduce the analyte fraction to a fine spray. In someembodiments, other ionization methods are used, such as inductivecoupled laser ionization, fast atom bombardment, soft laser desorption,atmospheric pressure chemical ionization, secondary ion massspectrometry, spark ionization, thermal ionization, and the like.

In some embodiments, the enriched fractions will be deposited on asurface for further analysis by matrix-assisted laserdesorption/ionization, surface enhanced laser desorption/ionization,immunoblot, and the like.

Some embodiments described herein relate to devices and methods forvisualizing an analyte in an electrophoretic separation before andduring the expulsion of enriched fractions.

Some embodiments described herein relate to devices and methods forvisualizing an analyte during an enrichment step.

Some embodiments described herein relate to devices and methods forvisualizing an analyte in a channel between enrichment zones.

In some embodiments, the visualization of an analyte can be performedvia optical detection, such as ultraviolet light absorbance, visiblelight absorbance, fluorescence, Fourier transform infrared spectroscopy,Fourier transform near infrared spectroscopy, Raman spectroscopy,optical spectroscopy, and the like.

Some embodiments described herein relate to devices that can enable theanalysis of analyte mixtures, in that they contain one or moreenrichment zones and an orifice to expel enriched analyte fractions. Insome embodiments, these devices include at least one layer which is nottransmissive to light of a specific wavelength, and at least one layerwhich is transmissive to that specific wavelength. One or more portionsof the layer which is not transmissive to light can define the one ormore enrichment zones, such that the enrichment zones serve as opticalslits.

In some embodiments, an analyte mixture can be loaded into a devicethrough a tube or capillary connecting the device to an autosampler. Insome embodiments, an analyte mixture can be loaded directly into areservoir on the device.

In some embodiments, an orifice through which at least a portion of asample can be expelled from a device is countersunk and/or shielded fromair flow. In some embodiments, this orifice is not electricallyconductive. As used herein, countersunk should be understood to meanthat a portion of a substrate defines a recess containing the orifice,irrespective of the geometry of the sides or chamfers of the recess.Similarly stated, countersunk should be understood to includecounterbores, conical and/or frustoconical countersinks, hemisphericalbores, and the like.

Some embodiments described herein relate to an apparatus, such as amicrofluidic device that includes a substrate constructed of an opaquematerial (e.g., soda lime glass, which is opaque to ultraviolet light).The substrate can define a microfluidic separation channel. Similarlystated, the microfluidic separation channel can be etched or otherwiseformed within the substrate. The microfluidic separation channel canhave a depth equal to the thickness of the substrate. Similarly stated,the microfluidic separation channel can be etched the full depth of thesubstrate (e.g., from the top all the way through to the bottom). Inthis way, the microfluidic separation channel can define an optical slitthrough the substrate. A transparent layer (e.g., a top layer) can bedisposed on a top surface of the substrate, for example, sealing the topsurface of the substrate. A transparent layer (e.g., a bottom layer) canalso be disposed on a bottom surface of the substrate, such that boththe top and the bottom of the microfluidic separation channel aresealed. In some embodiments, only a portion of the top layer and/or thebottom layer may be transparent. For example, the top layer and/or thebottom layer can define a transparent window in an otherwise opaquematerial; the window can provide optical access to, for example, themicrofluidic separation channel.

Some embodiments described herein relate to an apparatus, such as amicrofluidic device that includes a substrate. The substrate can defineone or more enrichment zones or channels. For example, the substrate candefine a first enrichment zone containing a media configured to bind toan analyte. Such a first enrichment zone can be suitable to separate ananalyte mixture chromatographically. The apparatus can further includetwo electrodes electrically coupled to opposite end portions of a secondenrichment zone. Such a second enrichment zone can be suitable toseparate an analyte mixture electrophoretically. The second enrichmentzone can intersect the first enrichment zone such that after a fractionof an analyte is separated, concentrated, and/or enriched in the firstenrichment zone, it can be further separated, concentrated, and/orenriched in the second enrichment zone. The device can also include arecessed orifice. The orifice can be an outlet of the second enrichmentchannel and can be disposed on a countersunk or otherwise recessedsurface of the substrate. The apparatus can be configured to expel aportion of an analyte mixture from the orifice via ESI. The recess canprovide a stable environment for formation of a Taylor cone associatedwith ESI and/or can be configured to accept an inlet port of a massspectrometer.

Some embodiments described herein relate to a method that includesintroducing an analyte mixture into a microfluidic device that containsa separation channel. An electric field can be applied across theseparation channel to effect a separation of the analyte mixture. Theanalyte mixture can be imaged during separation via a transparentportion of the microfluidic device. Similarly stated, a window and/oroptical slit can provide optical access to the separation channel suchthat the whole separation channel or a portion thereof can be imagedwhile the separation is occurring. A fraction of the analyte mixture canbe expelled from an orifice that is in fluid communication with theseparation channel. For example, the fraction can be expelled via ESI.In some embodiments, the orifice can be disposed on a countersunksurface of the microfluidic device such that a Taylor cone forms withina recess defined by the countersunk surface.

Some embodiments described herein relate to a method that includesinjecting an analyte into a microfluidic device containing a firstseparation channel and a second separation channel. The first separationchannel can contain a medium configured to bind an analyte from theanalyte mixture. Accordingly, when the analyte mixture is injected intothe microfluidic device at least a fraction of the analyte mixture canbe bound to the matrix and/or impeded from flowing through the firstseparation channel. For example, injecting the analyte into themicrofluidic device can effect a chromatographic separation in the firstseparation channel. An eluent can be injected into the microfluidicdevice such that at least a fraction of the analyte is mobilized fromthe media. The first separation channel can be imaged while the analyteis mobilized. Imaging the first separation can include whole column(e.g., whole channel) imaging and/or imaging a portion of the channel.An electric field can be applied to the second separation channel whenthe imaging detects that the fraction is disposed at an intersection ofthe first separation channel and the second separation channel such thatthe fraction is mobilized into the second separation channel. Forexample, in some embodiments, the first separation channel can beorthogonal to the second separation channel. Similarly stated the firstseparation channel and the first separation channel can form aT-junction. The imaging can detect when a portion of the fraction (e.g.,a portion of interest) is at the junction. Applying the electric fieldcan mobilize the portion of the fraction (and, optionally, not otherportions of the fraction that are not located at the junction) into thesecond separation channel for a second stage of separation. At least aportion of the fraction can be expelled from the microfluidic device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a device for two dimensionalseparation and ESI of an automatically loaded sample, according to anembodiment.

FIG. 2 is a schematic exploded view of a device having three layers,according to an embodiment.

FIG. 3 is a schematic of a light path through a microfluidic device,according to an embodiment.

FIG. 4 is a schematic illustration of a device for isoelectric focusing(IEF) and ESI of an automatically loaded sample, according to anembodiment.

FIG. 5 is a schematic illustration of a microfluidic device, accordingto an embodiment.

FIG. 6 is a flowchart of an exemplary method for analytecharacterization.

FIG. 7 is a schematic of a microfluidic device, according to anembodiment.

FIG. 8 is a schematic of a microfluidic device, according to anembodiment.

DETAILED DESCRIPTION OF INVENTION

It is to be understood that both the foregoing general description andthe following description are exemplary and explanatory only and are notrestrictive of the methods and devices described herein. In thisapplication, the use of the singular includes the plural unlessspecifically stated otherwise. Also, the use of “or” means “and/or”unless stated otherwise. Similarly, “comprise,” “comprises,”“comprising,” “include,” “includes” and “including” are not intended tobe limiting.

Devices

FIG. 1 is a schematic illustration of a device for two dimensionalseparation and ESI of an automatically loaded sample, according to anembodiment. A microfluidic network, 100, is defined by a substrate 102.The substrate is manufactured out of material which is compatible withthe enrichment steps being performed. For example, chemicalcompatibility, pH stability, temperature, transparency at variouswavelengths of light, mechanical strength, and the like are consideredin connection with selection of material.

Substrate 102 may be manufactured out of glass, quartz, fused silica,plastic, polycarbonate, polytetrafluoroethylene (PTFE),polydimethylsiloxane (PDMS), silicon, polyfluorinated polyethylene,polymethacrylate, cyclic olefin copolymer, cyclic olefin polymer,polyether ether ketone and/or any other suitable material. Mixtures ofmaterials can be utilized if different properties are desired indifferent layers of a planar substrate and/or any other suitablematerial. Mixtures of materials can be utilized if different propertiesare desired in different layers of a planar substrate.

Channels 106, 110, 114, 116, 118, 124 122, 126,132, 136 and 140 form themicrofluidic network 100 and are fabricated into substrate 102.Similarly stated, the substrate 102 defines channels 106, 110, 114, 116,118, 124 122, 126,132, 136 and/or 140.

Channels may be fabricated in the substrate through any channelfabrication method such as, for example, photolithographic etching,molding, machining, additive (3D) printing, and the like.

Analyte mixtures and external reagents can be loaded throughtube/conduit 112, and excess reagent/waste can be removed throughtube/conduit 130.

Tubes 112 and 130 can be manufactured out of any material compatiblewith the assay being performed, including, for example, fused silica,fused silica capillary tubes, silicone tubing, and/or PTFE tubing.

Channels 116 and 124 can be used to separate and/or enrich an analyteand/or a portion (e.g., a fraction) of an analyte. Channels 116 and/or124 can be used to perform chromatographic separations (e.g.,reversed-phase, immunoprecipitation, ion exchange, size exclusion,ligand affinity, dye affinity, hydrophobic interaction chromatography,hydrophilic interaction chromatography, pH gradient ion exchange,affinity, capillary electrokinetic chromatography, micellarelectrokinetic chromatography, high performance liquid chromatography(HPLC), amino acid analysis-HPLC, ultra performance liquidchromatography, peptide mapping HPLC, field flow fractionation—multiangle light scattering) or electrophoretic separations (e.g.,isoelectric focusing, capillary gel electrophoresis, capillary zoneelectrophoresis, isotachophoresis, capillary electrokineticchromatography, micellar electrokinetic chromatography, flowcounterbalanced capillary electrophoresis, electric field gradientfocusing, dynamic field gradient focusing). For example, channel 116 canbe derivatized or packed with material to perform a first enrichmentstep.

The material disposed into channel 116 and/or 124 can be selected tocapture analytes based on, for example, hydrophobicity (reversed-phase),immunoaffinity (immunoprecipitation), affinity (efficacy), size (sizeexclusion chromatography), charge (ion exchange) or by other forms ofliquid chromatography.

Many different methods can be used to dispose the enrichment materialwithin channels 116 and/or 124. The walls can be directly derivatizedwith, for example, covalently bound or adsorbed molecules, or beads,glass particles, sol-gel or the like can be derivatized and loaded intothese channels.

After sample is loaded into channel 116 wash solution and then elutionreagent can be introduced through tube 112 and channel 114.

The elution process will depend on the enrichment method performed inchannel 116. A suitable eluent can be selected to elute a fraction ofthe bound analyte. Some enrichment options may not require an elutionstep (e.g., size exclusion chromatography, electrophoretic separations,etc.).

The eluent or flow-through would then flow through channel 118 intochannel 124. Channel 124 could be used to perform either achromatographic or electrophoretic enrichment step.

Electrophoretic separations can be performed in channel 124 by using apower supply to apply an electric field between reservoir 108 andreservoir 120. Similarly stated, the device 100 can include electrodesin electrical contact with reservoir 108 and/or reservoir 120. Theelectrical ground of the power supply can be connected to the electricalground of a mass spectrometer to provide continuity in the electricfield from channel 124 to the mass spectrometer.

Any capillary electrophoresis (CE) electrophoretic method can beperformed in channel 124—IEF, isotachophoresis (ITP), capillary gelelectrophoresis (CGE), capillary zone electrophoresis (CZE), and thelike. Alternately, non-electrophoretic enrichment methods can beperformed in the channel 124.

In the case of IEF or ITP, concentrated purified sample bands would bemobilized, for example, by pressure or electrical means towardsconfluence 126. Sheath solution from reservoirs 108 and 134 could serveas sheath and catholyte.

The sheath/catholyte can be any basic solution compatible with theelectrophoretic separation and mass spectrometry (MeOH/N₄OH/H₂O forexample). Anolyte can be any acidic solution (e.g., phosphoric acid 10mM).

Alternately, the electric field could be reversed and catholyte (NaOH)could be loaded in reservoir 120, and anolyte could be used as thesheath solution in reservoirs 108 and 134.

The confluence 126 is where the enriched analyte fraction mixes with thesheath solution. As the analyte fractions in channel 124 are mobilized,solution will be pushed through confluence 126 out to orifice 128.

The orifice 128 can be disposed within a recess defined by surface 127of substrate 102. For example, surface 127 can be a countersunk ESIsurface. For example, as shown in FIG. 1, the enriched analyte solution,being electrically grounded through well 108, can form a Taylor coneemanating from orifice 128, which is disposed entirely within a recessdefined by surface 127. The orifice 128 and/or surface 127 can beoriented toward a mass spectrometer inlet, which can have a voltagepotential difference relative to well 108. As spray breaks off from thecone structure toward the mass spectrometer, it can be flanked bynebulizing gas provided through channels 106 and 140 before it leavesthe substrate 102. The nebulizing gas can be any inert or non-reactivegas (e.g., Argon, Nitrogen, and the like).

Additionally, using a sheath liquid and/or nebulizing gas can allow forthe use of an ion depleting step as the last “on-device” step. Thesheath liquid allows for replenishment of ion potential lost during anIEF charge assay concentrating step prior to ESI, and nebulizationprovides the sample in a fine mist for the off line analysis.

By generating the Taylor cone on surface 127, the cone is created in astable pocket or recess and is protected from disturbing air currents.Additionally, the conical geometry surrounding the countersunk orificehas a naturally expanding contact surface that will accommodate a widerrange of Taylor cone radial cross sections, allowing for a wider rangeof flow rates into the mass spectrometer.

Orifice 128 can be positioned in proximity to an inlet port of a massspectrometer. In some instances, the surface 127 can be configured suchthat an inlet port of a mass spectrometer can be disposed within arecess defined by the surface 127.

FIG. 2 a schematic exploded view of a device 212 having three layers,according to an embodiment. FIG. 2A shows a top layer 202 of device 212,according to an embodiment. FIG. 2B shows a middle layer 206 of device212, according to an embodiment. FIG. 2C shows a bottom layer 210 ofdevice 212, according to an embodiment. FIG. 2D shows the device 212 asassembled, according to an embodiment. Each of the three layers 202,206, 210 may be made of any material compatible with the assays thedevice 212 is intended to perform.

In some embodiments, layer 202 will be fabricated from a material whichis transparent to a specific wavelength, or wavelength range, of light.As used herein, “transparent” should be understood to mean that thematerial has sufficient transmittance to allow the amount of lighthaving a specific wavelength or range of wavelengths on one side of thematerial to be quantified by a detector on the other side. In someinstances, material with a transmissivity of 30%, 50%, 80%, 95%, or 100%is transparent. In some embodiments, a wavelength range of interest willinclude the middle ultraviolet range (e.g., 200 nm-300 nm), andmaterials such as, for example, glass, quartz, fused silica andUV-transparent plastics such as polycarbonates, polyfluorinatedpolyethylene, polymethacrylate, cyclic olefin polymer, cyclic olefincopolymer, and other UV-transparent materials can be used as transparentmaterials. In some embodiments, the light spectrum of interest will beexpanded beyond the visible spectrum (e.g., 200-900 nm).

Through-holes, 204, are fabricated in layer 202 to allow pressure andelectrical interface to a channel network in a lower layer (e.g., layer208) from outside the device.

FIG. 2B shows the internal middle layer 206 of device 212 containing thechannel network 208. The channel network is designed to interface withthe through-holes fabricated in the top layer 202. The channel network208 contains inlet and outlet tubes/conduits 209, and orifice 205 forexpelling enriched analyte fractions, and a viewable enrichment zone207. Enrichment zone 207 is fabricated so its depth is the fullthickness of the layer 206. In other embodiments, zone 207 can be lessthan the full thickness of layer 206.

In some embodiments, layer 206 will be fabricated from a material whichis opaque and/or not transparent to a specific wavelength, or wavelengthrange, of light. As used herein, “opaque” should be understood to meanthe material has insufficient transmittance to allow the amount of lighton one side of the material to be quantified by a detector on the otherside, and will effectively block this light except in the regions wherethe zone in the channel network is as deep as the full thickness oflayer 206.

FIG. 2C shows a bottom layer 210 of device 212. Bottom layer 210 can be,for example, a solid substrate. In some embodiments, bottom layer 210can be fabricated from a material with the same transmittance as layer202.

FIG. 2D shows the device 212 including top layer 202, the middle layer206, and the bottom layer 210, as assembled, according to an embodiment.Inlet and outlet tubes 209, reservoirs 204 and orifice 205 can still beaccessed after the device 210 is assembled. In some embodiments, theentire top layer 202 and/or the entire bottom layer 210 can betransparent. In other embodiments, a portion of the top layer 202 and/ora portion of the bottom layer 210 can be opaque with another portion ofthe top layer 202 and/or the bottom layer 210 being transparent. Forexample, the top layer 210 and/or the bottom layer 210 can define anoptical window that aligns with at least a portion of the enrichmentzone 207 when the device 212 is assembled.

FIG. 3 is a schematic of a light path through a microfluidic device 302,according to an embodiment. FIG. 3A shows a top view of the microfluidicdevice 302. FIG. 3B shows the microfluidic device 302 positioned betweena light source 306 and a detector 308. The detector 308 is positioned tomeasure light passing through the device 302. While not illustrated inFIG. 3, the microfluidic device 302 can have a similar channel structureas described in FIGS. 1 and 2, but the channel structure is not shownfor ease of reference. In some embodiments, a portion of top surface ofthe microfluidic device 302 is opaque and completely or substantiallyobscures light projected from the light source 306 from reaching thedetector 308. The portion of the opaque top surface substantiallyprevents the transmission of light through the device at those portionswhere detection of sample properties is not desired. For example, themicrofluidic device 302 in some embodiments is not opaque (e.g., allowssome light to pass through) over one or more channel region(s) 304, asthe channel 304 transverses the entire thickness of a non-transparentlayer.

In some embodiments, this transparent channel region(s) 304, can be anenrichment zone, where optical detection can be used to detect analyte,monitor the progress of the enrichment and/or monitor enriched analytefraction(s) as they are expelled from the device. In some embodiments,changes in the amount of light passing through transparent channel 304will be used to measure the absorbance of the analyte fractions whilethey are in this channel. Thus, in some embodiments, channel region(s)304 define an optical slit, such that the light source 306 positioned onone side of the microfluidic device 302 effectively illuminates thedetector 308 only through the transparent channel region(s) 304. In thisway, stray light (e.g., light that does not pass thorough thetransparent channel regions(s) and/or a sample) can be effectivelyblocked from the detector 308, which can reduce noise and improve theability of the detector 308 to observe sample within the transparentchannel region(s) 304. In some embodiments, the transparent channelregions(s) 304 will be between two enrichment zones, and can be used todetect analyte fractions as they are eluted from the upstream enrichmentzone.

Methods

FIG. 6 illustrates a method of analyte mixture enrichment according toan embodiment. The method includes loading and/or introducing an analytemixture onto a microfluidic device, at 20. The microfluidic device canbe similar to the microfluidic devices described above with reference toFIGS. 1-3. In some embodiments, the analyte mixture can be, for example,glycans, carbohydrates, DNA, RNA, intact proteins, digested proteins,peptides, metabolites, vaccines, viruses and small molecules. In someembodiments, the analyte mixture can be a mixture of proteins, such as alysate of cultured cells, cell-based therapeutics, or tumor or othertissue derived cells, recombinant proteins, including biologicpharmaceuticals, blood derived cells, perfusion or a protein mixturefrom any other source. The analyte mixture may be loaded directly ontothe device, or may be loaded onto an autosampler for serial analysis ofmultiple mixtures.

The microfluidic device can include a first separation channel and/orenrichment zone. In some embodiments, the first separation channeland/or enrichment zone can be configured for chromatographic separation.For example, the first separation channel and/or enrichment zone cancontain a media configured to bind an analyte from the analyte mixtureand/or otherwise effect a chromatographic separation. At 21, a firstenrichment can be performed; for example, a chromatographic separationcan be performed in the first separation channel and/or enrichment zone.In some embodiments, such as embodiments in which the analyte mixture isa protein mixture, the first enrichment, at 21, can simplify the proteinmixture. The first enrichment, at 21, can be based on any discernablequality of the analyte.

This enriched analyte fraction is then eluted, at 22. For example, aneluent can be injected into the microfluidic device to mobilize theenriched analyte fraction from media disposed within the firstseparation channel and/or enrichment zone. In some embodiments, theenrichment and/or mobilization of the enriched analyte fraction can beimaged. For example, as discussed above, the first separation channeland/or enrichment zone can define an optical slit. Light can beprojected onto the microfluidic device and a detector can detect lightpassing through the first separation channel and/or enrichment zone. Thesample, or a portion thereof can be detected via absorbance and/orfluorescence imaging techniques.

The microfluidic device can include a second separation channel and/orenrichment zone. In some embodiments, the second separation channeland/or enrichment zone can be configured for electrophoretic separation.At 23, a second enrichment can be performed, for example, on the eluate.For example, an electric field and/or electric potential can be appliedacross the second separation channel and/or enrichment zone.

In some embodiments, the second enrichment can be initiated, at 23, whena fraction of the analyte mixture is disposed at an intersection of thefirst separation channel and/or enrichment zone and the secondseparation channel and/or enrichment zone. For example, the firstseparation channel and/or enrichment zone can be monitored (e.g.,imaged) and an electric potential, and/or electric field can be appliedwhen a fraction of interest reaches the intersection.

In some embodiments, the second enrichment, at 23, can provide fractionsenriched based on charge characteristics (charge isoforms). Suchenrichments can include, for example, gel isoelectric focusing,isoelectric focusing with mobilization, isoelectric focusing with wholecolumn imaging, ion exchange chromatography, pH gradient exchangechromatography, isotachophoresis, capillary zone electrophoresis,capillary gel electrophoresis or other enrichment techniques that are,for example, charge-based.

Although the first enrichment, at 21, has been described as achromatographic enrichment and the second enrichment, at 23, has beendescribed as electrophoretic, it should be understood the any suitableenrichment can be performed in any suitable sequence. For example, thefirst enrichment, at 21, and the second enrichment, at 23, can both bechromatographic or both be electrophoretic. As another example, thefirst enrichment, at 21, can be electrophoretic, and the secondenrichment, at 23, can be chromatographic.

In some embodiments, one or more enrichments can provide fractionsenriched based on hydrophobic changes, such as oxidation. Suchenrichments can include, for example, reversed-phase chromatography,hydrophobic interaction chromatography, hydrophilic interactionchromatography, or other enrichment techniques that are, for example,hydrophobicity-based.

In some embodiments, one or more enrichments can provide fractionsenriched based on post-translational modifications, glycoforms includinggalactosylation, fucosylation, sialylation, mannose derivatives andother glycosylations, as well as glycation, oxidation, reduction,phosphorylation, sulphanation, disulfide bond formation, deamidiation,acylation, pegylation, cleavage, antibody-drug conjugation (ADC),protein-drug conjugation, C-terminal lysine processing, other naturallyand non-naturally occurring post-translational modifications and otherchemical and structural modifications introduced after translation ofthe protein, and the like. Such enrichments can include, for example,binding assays and the like.

In some embodiments, one or more enrichments can provide fractionsenriched based on hydrophobic changes, such as oxidation. Suchenrichments can include, for example, reversed-phase chromatography,hydrophobic interaction chromatography, hydrophilic interactionchromatography, or other enrichment techniques that arehydrophobicity-based.

In some embodiments, one or more enrichments can provide fractionsenriched based on primary amino acid sequence, such as caused bymutation, amino acid substitution during manufacture and the like. Suchenrichments can include, for example, separating by charge isoforms,hydrophobic changes, or other enrichment techniques that can distinguishbetween primary amino acid sequence differences.

In some embodiments, one or more enrichments can provide fractionsenriched based on efficacy. Such enrichments can include, for example,bioassays, enzyme inhibition assays, enzyme activation assays,competition assays, fluorescence polarization assays, scintillationproximity assays, or other enrichment techniques that are efficacy-basedand the like.

In some embodiments, one or more enrichments can provide fractionsenriched based on affinity. Such enrichments can include, for example,solution phase binding to target, binding to bead based targets, surfacebound target, immunoprecipitation, protein A binding, protein G bindingand the like.

In some embodiments, one or more enrichments can provide fractionsenriched based on mass or size. Such enrichments can include, forexample, poly acrylamide gel electrophoresis, capillary gelelectrophoresis, size exclusion chromatography, gel permeationchromatography, or other enrichment techniques that are mass-based.

In some embodiments, the analyte mixture will go through more than twoenrichments and/or enrichment channels before being expelled from thedevice.

At 24, an enriched analyte fraction can be expelled from the device. Insome embodiments, the enriched analyte fraction can be expelled via IEF.Expelling the enriched analyte fraction, at 24, can concentrate theanalyte fractions before they are expelled from.

In some embodiments the analyte fractions are expelled, at 24, using anionization technique, such as electrospray ionization, atmosphericpressure chemical ionization, and the like.

In some embodiments, the analyte fractions are expelled, at 24, usingelectrokinetic or hydrodynamic forces.

In some embodiments, the enriched protein fractions are expelled, at 24,from the device in a manner coupled to a mass spectrometer.

Mass of an analyte expelled from the microfluidic device (e.g., abiologic or biosimilar) can be measured, for example, throughtime-of-flight mass spectrometry, quadrupole mass spectrometry, Ion trapor orbitrap mass spectrometry, distance-of-flight mass spectrometry,Fourier transform ion cyclotron resonance, resonance mass measurement,and nanomechanical mass spectrometry.

In some embodiments pI markers are used to map pI ranges in thevisualized IEF channel (e.g., the first separation channel and/orenrichment zone and/or the second separation channel and/or enrichmentzone). In some embodiments, pI markers or ampholytes can be used todetermine the pI of the analyte by their presence in downstream massspectrometry data.

In some embodiments, IEF can be monitored during the mobilization andESI. In this way, mass spectrometry data can be correlated to peaks inthe IEF, which can maintain and/or improve peak resolution.

In some embodiments, the analyte mixture and/or a portion thereof can bemobilized within the microfluidic device using pressure source. In someembodiments, mobilization is done with hydrostatic pressure. In someembodiments, mobilization is chemical immobilization. In someembodiments, mobilization is electrokinetic mobilization

FIG. 7 is a schematic of a microfluidic device, according to anembodiment. A microfluidic network, 800, is disposed in and/or definedby a substrate, 802. The substrate is manufactured out of material whichis compatible with the enrichment steps being performed. For example,chemical compatibility, pH stability, temperature, transparency atvarious wavelengths of light, mechanical strength, and the like may beof concern when selecting the material

Substrate 802 may be manufactured out of glass, quartz, fused silica,plastic, polycarbonate, PTFE, PDMS, silicon, polyfluorinatedpolyethylene, polymethacrylate, cyclic olefin copolymer, cyclic olefinpolymer, polyether ether ketone and/or any other suitable material.Mixtures of materials can be utilized if different properties aredesired in different layers of a planar substrate.

Channels 806, 808, 810, 811, 817, 814, 812 form a channel network andare fabricated into (e.g., defined by) substrate 802.

Channels may be fabricated in the substrate through any channelfabrication method such as photolithographic etching, molding,machining, additive (3D) printing, and the like.

Analyte mixtures and external reagents can be loaded through tube 804,and excess reagent/waste can be removed through tube 810 and 818.

Tubes 804 810, and/or 818 can be manufactured out of any materialcompatible with the assay being performed, including fused silica, fusedsilica capillary tubes, silicone tubing, PTFE tubing, and the like.

Channels 806 and 814 can be designated as separation/enrichment zones.Either of channel 806 and/or 814 can be used to perform chromatographicseparations (reversed phase, immunoprecipitation, ion exchange, sizeexclusion, ligand affinity, dye affinity, hydrophobic interaction,affinity, capillary electrokinetic chromatography, micellarelectrokinetic chromatography and/or the like) or electrophoreticseparations (isoelectric focusing, capillary gel electrophoresis,capillary zone electrophoresis, isotachophoresis, capillaryelectrokinetic chromatography, micellar electrokinetic chromatography,flow counterbalanced capillary electrophoresis, electric field gradientfocusing, dynamic field gradient focusing, and/or the like). Forexample, channel 806 can be derivatized or packed with material toperform a first enrichment step, represented by darker circles inchannel 806.

The material disposed into channel 806 can be selected to captureanalytes based on hydrophobicity (reversed phase), affinity (efficacy),size (size exclusion chromatography), charge (ion exchange),immunoaffinity (immunoprecipitation), protein-protein interaction,DNA-protein interaction, aptamer-base capture, small molecule-basecapture or by other forms of liquid chromatography and the like.

Many different methods can be used to dispose the enrichment materialwithin channel 806 and/or 814. The walls can be directly derivatizedwith covalently bound or adsorbed molecules, or beads, glass particles,sol-gel or the like can be derivatized and loaded into these channels,or channels can be packed with a sieving material such as—linear polymersolutions such as linear polyacrylamide (LPA), polyvinylpyrrolidone(PVP), polyethylene oxide (PEO), dextran, and the like, cross-linkedpolymer solutions such as polyacrylamide and the like, matrices forliquid chromatography, or other materials.

Chemically reactive solutions may be added depending on the particularassay performed. In some cases, derivatization of material may occurafter it is loaded into channel 806 (or channel 814), by addingmolecules which will adsorb or covalently bond to the loaded material,or can chemically cross link reactive elements to the material. Forexample, material coated with an antibody-binding molecule such asprotein A, protein G, epoxy or the like, could be disposed into channel806. Subsequent rinsing with an antibody solution would leave thematerial coated with antibody and able to participate in immunoaffinitycapture. In some cases, the antibody may be mixed with a target analyteor lysate so that the antibody can bind its target in free solutionbefore being coated onto the material.

After enrichment materials are loaded onto device, sample is loaded viatube 804 into channel 806. Subsequently, wash solutions and elutionreagents can be introduced through tube 804 to channel 806.

In some cases, detection reagents will be added to bind to capturedmaterial. Numerous labeling reagents are available that can covalentlyattach detection moieties such as fluorophores, chromophores or otherdetection molecules to the target proteins at terminal ends of thepolypeptide, and by attachment to amino acid side chains such as lysine,cysteine and other amino acid moieties. Covalently bound detectionmoieties allow for the protein to be detected through fluorescenceexcitation, chromophoric assay, or other indirect means. In some cases,the target protein can remain unlabeled and detected through nativeabsorbance at 220nm, 280nm or any other wavelength at which the proteinwill absorb light, or native fluorescence. In some cases, the proteinwill be detected using non-covalently bound fluorogenic, chromogenic,fluorescent or chromophoric labels, such as SYPRO® ruby, Coomassie blueand the like.

In some cases, detection reagents will be added directly to channel 814to aid detection.

The elution process will depend on the enrichment method performed inchannel 806. It will be selected to elute at least a fraction of thebound analyte. In some cases, this can be accomplished with acombination of heat and sodium dodecyl sulfate (SDS), or otherdetergents, glycine, urea, or any other method which will induce therelease of the captured analyte. Some enrichment options may not requirea direct elution step (e.g. size exclusion chromatography). In somecases, elution will be followed by denaturation.

The eluent would then flow through channel 808 into the nextseparation/enrichment zone, channel 814. Channel 814 could be used toperform either a chromatographic or electrophoretic enrichment step.

Electrophoretic separations can be performed in channel 814 by using apower supply to apply an electric field between reservoir 812 andreservoir 816. When eluate from channel 806 passes through theintersection of channels 808 and 814, the electric field can be enabled,loading analyte into channel 814. In some case, the analyte will benegatively charged, such as in the standard gel electrophoresis modewhere protein analyte is saturated with a negatively charged detergentlike SDS. However, the polarity of channel 814 can easily be reversed toaccommodate systems where for example, a protein analyte is saturatedwith a positively charged detergent such as cetyl trimethylammoniumbromide (CTAB) or the like. In other cases, a protein analyte may becoated with a neutral detergent, or no detergent—such as in native gelelectrophoresis. In this case, polarity will be selected based on theanticipated charge of the protein target in the buffer system selected,so that the protein analyte will migrate into channel 814.

Any CE electrophoretic method can be performed in channel 814—IEF, ITP,CGE, CZE, and the like. Alternately, non-electrophoretic enrichmentmethods can be performed in the channel.

Analyte in channel 814 can be viewed by whole column imaging, partialcolumn imaging, and/or by single point detection.

In some cases, the enrichment material in channels 806, 814 or both maybe removed and replenished with fresh material so that the device can beused on another analyte sample.

In some cases, a channel design such as FIG. 7 may be repeated multipletimes on a device, so that more than one analyte sample may be analyzedin parallel.

EXAMPLES

Aspects of embodiments may be further understood in light of thefollowing examples, which should not be construed as limiting in anyway.

Example 1 Characterize Protein Charge on Chip Before Mass Spectrometry(MS)

For this example, the channel network shown in FIG. 4 is fabricated froma plate of soda lime glass, which has very low transmission of 280nmlight using a standard photolithographic etching technique. The depth ofthe enrichment channel 418 is the same as the thickness of the glasslayer 402, i.e., the enrichment channel 418 passes all the way from thetop to bottom of this glass plate 402. The device 400 can be illuminatedby a light source disposed on one side of device 400 and imaged by adetector on disposed on an opposite side of device 400. Becausesubstrate 402 is opaque, but enrichment channel 418 defines an opticalslit, the substrate 402 can block light that does not pass through theenrichment channel 418, blocking stray light and improving resolution ofthe imaging process.

The glass layer 402 is sandwiched between two fused silica plates, whichare transmissive (e.g., transparent) to 280nm light. As in FIG. 2, thetop plate contains through holes for the instrument and user tointerface with the channel network, while the bottom plate is solid. The3 plates are bonded together at 520° C. for 30 minutes. The inlet andoutlet tubing is manufactured from cleaved capillary (100 μm ID,polymicro), bonded to the channel network.

The device is mounted on an instrument containing a nitrogen gas source,heater, positive pressure pump (e.g., Parker, T5-11C-03-1EEP),electrophoresis power supply (Gamm High Voltage, MC30) terminating intwo platinum-iridium electrodes (e.g., Sigma-Aldrich, 357383), UV lightsource (e.g., LED, qphotonics, UVTOP280), CCD camera (e.g., ThorLabs,340UV-GE) and an autosampler for loading samples onto the device. Thepower supply shares a common earth ground with the mass spectrometer.The instrument is controlled through software (e.g., labView).

Protein samples are pre-mixed with ampholyte pH gradient and pI markersbefore placing into vials and loading onto the autosampler. They areserially loaded from an autosampler via the inlet 412 onto themicrofluidic device 400 through the enrichment channel 418 and out ofthe device to waste 430 through the outlet 434.

The sheath/catholyte fluid (50% MeOH, N₄OH/H₂O) is loaded onto the twocatholyte wells 404, 436, anolyte (10 mM H₃PO₄) onto the anolyte well426, and the source of heated nitrogen gas is attached to the two gaswells 408, 440.

After all reagents are loaded, an electric field of +600V/cm is appliedfrom anolyte well 426 to catholyte wells 404, 436 by connecting theelectrodes to the anolyte well 426 and catholyte wells 404, 436 toinitiate isoelectric focusing. The UV light source is aligned under theenrichment channel 418, and the camera is placed above the enrichmentchannel 418 to measure the light that passes through the enrichmentchannel 418, thereby detecting the focusing proteins by means of theirabsorbance. The glass plate 402, being constructed of soda-lime glass,acts to block any stray light from the camera, so light not passingthrough the enrichment channel 418 is inhibited from reaching thecamera, increasing sensitivity of the measurement.

Images of the focusing proteins can be captured continuously and/orperiodically during IEF. When focusing is complete, low pressure will beapplied from the inlet 412, mobilizing the pH gradient toward theorifice 424. The electric field can be maintained at this time tomaintain the high resolution IEF separation. Continuing to image theenrichment channel 418 during the ESI process can be used to determinethe pI of each protein as it is expelled from the orifice 424.

As the enriched protein fraction moves from the enrichment channel 418into the confluence 420, it will mix with the sheath fluid, which canflow from the catholyte wells 404, 436 to the confluence 420 viasheath/catholyte fluid channels 406, 438. Mixing enriched proteinfractions with the sheath fluid can put the protein fraction in a massspectrometry compatible solution, and restore charge to the focusedprotein (IEF drives proteins to an uncharged state), improving theionization.

The enriched protein fraction then continues on to the orifice 424,which can be defined by a countersunk surface 422 of the glass plate402. The enriched protein fraction can create a Taylor cone once caughtin the electric field between the sheath fluid well ground and massspectrometer negative pole.

As solution continues to push at the Taylor cone from the enrichmentchannel 418, small droplets of fluid will be expelled from the Taylorcone and fly towards the mass spectrometer inlet. Nitrogen gas (e.g., at150° C.) can flow from the gas wells 408, 440, down gas channels 410,432 and form nitrogen gas jets which flank the Taylor cone which canconvert droplets emanating from the Taylor cone to a fine mist beforeleaving the microfluidic device, which can aid detection in the massspectrometer. Adjusting pressure from the inlet 412 can adapt Taylorcone size as needed to improve detection in mass spectrometer.

Example 2 Reversed-Phase→IEF→MS

Example 2 can be similar to example 1, but is described with referenceto FIG. 1. The channel 116 can be a first enrichment zone loaded withsol-gel derivatized with C18. After loading protein, a volume of eluent(MeCN/H₂O with IEF ampholytes and standards) can be loaded into channel116 to elute the least hydrophobic proteins trapped on the sol gel. Theeluate is directed to channel 124, which can be a second enrichment zonewhere IEF, UV absorbance monitoring and finally ESI take place asdescribed in example 1. Once the ESI of the first eluate is complete, avolume of higher MeCN concentration is used to elute the next lowesthydrophobic protein fraction.

Example 3 Efficacy→IEF→MS

Example 3 can be similar to example 2, but biologic drug targetderivatized beads can be loaded into channel 116 and used to captureprotein. Affinity of reaction is characterized through elution bysolution phase target (competitive), salt, pH, or the like.

Example 4 Reversed-Phase→Capillary Zone Electrophoresis→MS

Example 4 can be similar to example 2, but is described with referenceto FIG. 5. A protein mixture can be loaded through inlet 521 and passthrough to enrichment zone 510, which can contain beads derivatized withC18 for reversed-phase chromatography. During loading, fluid passesthrough the zone 510, through viewing region 511 and out outlet 522 towaste. Viewing region 510 transverses an internal layer made ofsoda-lime glass, which is opaque to 280 nm UV light, while the top andbottom layers are made of fused silica, which are transparent to 280 nmlight.

A 280 nm light source is positioned below viewing region 511 and a CCDdetector is placed above viewing region 511.

A solution of 20% MeCN/H₂O is loaded through inlet 521 throughenrichment zone 510. This solution will elute a fraction enriched forthe least hydrophobic proteins in the mixture. Viewing region 511 ismonitored for the absorbance of the enriched protein fraction at 280nmas it moves from enrichment zone 510 to the outlet 522. When thefraction is positioned at the intersection of enrichment zone 510 andenrichment zone 515, a power supply is turned on creating an electricfield between a positive electrode in reservoir 514 and ground atreservoir 504. This polarity can easily be reversed by switching thepolarity of the power supply. Once the electric field is present, theenriched protein fraction will migrate down enrichment zone 515separating proteins by capillary zone electrophoresis. The separatedproteins will mix with the sheath, electrolyte solution at confluence516, and form a Taylor cone on surface 518. Nebulizing Nitrogen gas lineis connected to the device at ports 508 and 528, and moves throughchannels 512 and 530 to flank material from the electrospray as it exitsthe device via orifice 520.

Alternatively, hydrodynamic pressure could be used to load the enrichedprotein fraction into enrichment zone 515.

Example 5 Immunoprecipitation→Capillary Gel Electrophoresis of ProteinLysates

In this example, a microfluidic channel layer represented by the layoutin FIG. 7 is fabricated from a cyclic olefin copolymer. Similarlystated, substrate 802 of microfluidic device 800 defines a channelnetwork. For many applications, for example, if fluorescent detection isemployed, microfluidic device 800 could be manufactured using a singlematerial, provided that this material will transmit the wavelength rangeof light needed to detect the analyte.

Protein A coated beads are loaded into channel 806. These beads arerinsed with a solution of antibody raised against a target of interest,which will bind to the protein A beads. To reduce antibody sheddinginterfering with analyte detection, the antibody is then covalentlycross-linked to the antibody to the bead using commercially availablecross linking reagents, such as Dimethyl pimelimidate (DMP),Bis(sulfosuccinimidyl)suberate (BS3) and the like. Afterimmunoprecipitation beads are prepared and loaded in channel 806, lysateanalyte sample can be loaded via tube 804. After analyte is givensufficient time to be captured by immobilized antibody, unbound proteinsare washed and cleared to waste via tube 822.

Next, the protein is eluted from the antibody beads so it can beanalyzed. Elution is accomplished by loading solution of sodium dodecylsulfate (SDS) and heating to 50C for 10 minutes. Once released, theeluted analyte is flowed through channel 808 toward the intersection ofchannel 808 and 814. When the analyte plug reaches the intersection ofchannel 808 and 814, an electric field is turned on between a negativepole at reservoir 812 and a positive pole at reservoir 816, causing thenegatively charged protein to migrate through a dextran linear polymersolution in channel 814, which has been loaded with the fluorogenicprotein dye SYPRO® ruby.

Fluorescently labeled target protein can be visualized during CGE inchannel 814 using whole column imaging. Similarly stated, the entiretyof channel 814 can be imaged while the SYPRO® ruby dye is excited with280nm light and emitted light, at 618nm, is measured by a detector.

Example 6 Variations of Microfluidic Design without Mass SpectrometerInterface

In some cases, it will be advantageous to have two designs of amicrofluidic layer, that differ by presence or absence of the massspectrometer interface. Once an analyte is characterized, confirmatorycharacterization may be done in the absence of the mass spectrometrydata. By doing the confirmatory characterization in nearly the samemicrofluidic design, when an anomaly is identified, it will be simple totransfer the assay back to the chip with the mass spec interface formass identification. This can eliminate the work otherwise needed toshow that the anomaly in the confirmatory data is being analyzed in themass spectrometry data.

As an example, FIG. 8 shows a microfluidic design similar tomicrofluidic device 400 shown in FIG. 4, without orifice 424 andcountersunk surface 422. Analyte is still introduced to the chip throughan inlet 904 and channel 906 to an enrichment channel 908, but afteranalysis the sample will be flushed out through an outlet channel 910,rather than conducting electrospray ionization at an orifice. Thisdesign could be run for general operation, and then at times when massidentification is required, the same enrichment can be performed on themicrofluidic device 400, shown in FIG. 4, ensuring identification of theanalyte variants see on microfluidic device 900 of FIG. 8.

The foregoing descriptions of specific embodiments of the invention havebeen presented for purposes of illustration and description. They arenot intended to be exhaustive or to limit the invention to the preciseforms disclosed, and obviously many modifications and variations arepossible in light of the above teaching. Although various embodimentshave been described as having particular features and/or combinations ofcomponents, other embodiments are possible having a combination of anyfeatures and/or components from any of embodiments where appropriate.The embodiments were chosen and described in order to best explain theprinciples of the invention and its practical application, to therebyenable others skilled in the art to best utilize the invention andvarious embodiments with various modifications as are suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto and theirequivalents.

Where methods and/or schematics described above indicate certain eventsand/or flow patterns occurring in certain order, the ordering of certainevents and/or flow patterns may be modified. Additionally certain eventsmay be performed concurrently in parallel processes when possible, aswell as performed sequentially. While the embodiments have beenparticularly shown and described, it will be understood that variouschanges in form and details may be made.

All patents, patent applications, publications, and references citedherein are expressly incorporated by reference to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. A method, comprising: (a) applying a firstelectric field across a first fluid channel in a microfluidic device toseparate a mixture of analytes and ampholytes via isoelectric focusing;(b) applying a second electric field across the first fluid channel tomobilize the separated analytes; and (c) expelling the mobilizedanalytes via electrospray ionization from an orifice in the microfluidicdevice into a mass spectrometer; wherein imaging of the first fluidchannel or a portion thereof is used to monitor the separation in (a)and the mobilization in (b).
 2. The method of claim 1, wherein themixture of analytes comprises a mixture of intact proteins.
 3. Themethod of claim 1, wherein the microfluidic device comprises an opticalslit that provides optical access to the first fluid channel, andwherein the imaging comprises detecting light that has passed through oris emitted from the first fluid channel.
 4. The method of claim 1,wherein the imaging comprises absorbance imaging to detect and monitorseparated analyte peaks.
 5. The method of claim 1, wherein the imagingcomprises fluorescence imaging to detect and monitor separated analytepeaks.
 6. The method of claim 5, wherein the fluorescence imagingcomprises imaging of native fluorescence.
 7. The method of claim 1,further comprising introducing isoelectric point (pI) markers into thefirst fluid channel prior to performing the isoelectric focusing in (a),and wherein the imaging further comprises detection and monitoring ofpositions of the pI markers.
 8. The method of claim 7, wherein thepositions of the pI markers are used to determine an isoelectric point(pI) for one or more separated analytes.
 9. The method of claim 1,further comprising correlating separated analyte peaks detected in thefirst fluid channel with mass spectrometer data for the separatedanalytes.
 10. The method of claim 1, wherein the microfluidic devicefurther comprises a nebulizing gas delivery channel for facilitating theelectrospray ionization.
 11. The method of claim 1, wherein themicrofluidic device further comprises a second fluid channel that is influid communication with an end of the first fluid channel that isopposite an end that is in fluid communication with the orifice.
 12. Themethod of claim 11, wherein a chromatographic enrichment is performed inthe second fluid channel prior to performing the isoelectric focusingseparation in (a).
 13. The method of claim 12, wherein thechromatographic enrichment comprises a reversed-phase,immunoprecipitation, ion exchange, size exclusion, or affinitychromatographic enrichment.
 14. A method, comprising: (a) separating amixture of analytes in a first fluid channel in a microfluidic device;(b) mobilizing the separated analytes in the first fluid channel; and(c) expelling the mobilized analytes via electrospray ionization from anorifice in the microfluidic device into a mass spectrometer; whereinimaging of the first fluid channel or a portion thereof is used tomonitor the separation in (a) and the mobilization in (b).
 15. Themethod of claim 14, wherein the separation in (a) is performed usingisoelectric focusing or capillary electrophoresis.
 16. The method ofclaim 14, wherein the separation in (a) is performed usingchromatography.
 17. The method of claim 14, wherein the analyte mixturecomprises proteins.
 18. The method of claim 17, wherein the proteinscomprise intact proteins.
 19. The method of claim 14, wherein theimaging comprises absorbance imaging to detect and monitor separatedanalyte peaks.
 20. The method of claim 14, wherein the imaging comprisesfluorescence imaging to detect and monitor separated analyte peaks. 21.The method of claim 20, wherein the fluorescence imaging comprisesimaging of native fluorescence.
 22. The method of claim 14, wherein themobilization of separated analytes in (b) is performed by introducing aneluent into the first fluid channel using pressure.
 23. The method ofclaim 14, wherein the mobilization of separated analytes in (b)comprises chemical mobilization or electrokinetic mobilization.
 24. Themethod of claim 14, wherein the microfluidic device comprises an opticalslit that provides optical access to the first fluid channel, andwherein the imaging comprises detecting light that has passed through oris emitted from the first fluid channel.
 25. The method of claim 14,further comprising introducing isoelectric point (pI) markers into thefirst fluid channel prior to performing the isoelectric focusing in (a),and wherein the imaging further comprises detection and monitoring ofpositions of the pI markers.
 26. The method of claim 25, wherein thepositions of the pI markers are used to determine an isoelectric point(pI) for one or more of the separated analytes.
 27. The method of claim14, wherein the microfluidic device further comprises a second fluidchannel that is in fluid communication with an end of the first fluidchannel that is opposite an end of the first fluid channel that is influid communication with the orifice, and wherein a chromatographicenrichment is performed in the second fluid channel prior to performingthe separation in (a).
 28. The method of claim 27, wherein thechromatographic enrichment comprises a reversed-phase,immunoprecipitation, ion exchange, size exclusion, or affinitychromatographic enrichment.
 29. The method of claim 14, furthercomprising correlating separated analyte peaks detected in the firstfluid channel with mass spectrometer data for the separated analytes.30. The method of claim 14, wherein the microfluidic device furthercomprises a nebulizing gas delivery channel for facilitating theelectrospray ionization.